This invention relates to therapies, diagnostics, and research reagents for disease states which respond to modulation of the expression of the xcex2I, xcex2II, xcex3, xcex4, xcex5, xcex6 or xcex7 isoform of protein kinase C. In particular, preferred embodiments, this invention relates to sequence-specific antisense phosphorothioate oligonucleotides comprising nucleosides joined by intersugar linkages, wherein the intersugar linkages are substantially pure (i.e. substantially all Sp or Rp) chiral phosphorothioate linkages which are specifically hybridizable with nucleic acids relating to the xcex2I, xcex2II, xcex3, xcex4, xcex5, xcex6 and xcex7 isoforms of protein kinase C. These oligonucleotides modulate the expression of the xcex2I, xcex2II, xcex3, xcex4, xcex5, xcex6 and xcex7 isoforms of protein kinase C, and are especially well suited as diagnostics, therapeutics and research reagents.
1. Protein Kinase C Isoforms
The phosphorylation of proteins plays a key role in the transduction of extracellular signals into cells. Kinases, the enzymes which effect such phosphorylations, are targets for the action of growth factors, hormones, and other agents involved in cellular metabolism, proliferation and differentiation. One of the major signal transduction pathways involves the enzyme protein kinase C (PKC), which is known to have many critical influences on cell proliferation and differentiation. PKC is activated by diacylglycerols (DAGs), which are metabolites released in signal transduction.
PKC is not a single enzyme, but a family of enzymes. At the present time at least seven isoforms (isozymes) of PKC have been identified. Isoforms xcex1, xcex2, and xcex3 have been purified to homogeneity and isoforms xcex4, xcex5, xcex6 and xcex7 have been identified by molecular cloning. These isozymes have distinct patterns of tissue and organ localization (see Nishizuka, Nature, 334:661, 1988, for a review) and may serve different physiological functions. For example, PKC-xcex3 seems to be expressed only in the central nervous system. As another example, PKC-xcex7 has been found predominantly in the skin and lungs, with levels of expression much higher in these tissues than, for example, in the brain. This is in contrast to other members of the PKC family which tend to be most abundantly expressed in the brain (Osada et al., J. Biol. Chem. 265:22434, 1990). As a third example, PKC-xcex1 and -xcex2 are expressed in most tissues, but have different patterns of expression in different cell types. Both PKC-xcex1 and PKC-xcex2 are expressed in, and have been purified from, human epidermis; however, while PKC-xcex1 has been detected mainly in keratinocytes of the basal layers of the epidermis, PKC-xcex2 is found mainly in the middle layers of the epidermis and Langerhans cells. It is presently believed that different PKC isozymes may be involved in various disease processes depending on the organ or tissue in which they are expressed. For example, in psoriatic lesions there is an alteration in the ratio between PKC-xcex1 and PKC-xcex2, with preferential loss of PKC-xcex2 compared to normal skin (Hegemann, L. and G. Mahrle, Pharmacology of the Skin, H. Mukhtar, ed., pp. 357-368, CRC Press, Boca Raton, Fla., 1992).
Interest in PKC was stimulated by the finding that PKC is a major cellular receptor through which a class of tumor-promoting agents called phorbol esters exert their pleiotropic effects on cells (Gescher et al., Anti-Cancer Drug Design 4:93, 1989). Phorbols capable of tumor production can mimic the effect of DAG in activating PKC, suggesting that these tumor promoters act through PKC and that activation of PKC is at least partially responsible for the resulting tumorigenesis (Parker et al., Science 233:853, 1986).
Experimental evidence indicates that PKC plays a role in growth control in colon cancer. It is believed that specific bacteria in the intestinal tract convert lipids to DAG, thus activating PKC and altering cell proliferation. This may explain the correlation between high dietary fat and colon cancer (Weinstein, Cancer Res. (Suppl.) 51:5080s, 1991). It has also been demonstrated that a greater proportion of the PKC in the colonic mucosa of patients with colorectal cancer is in an activated state compared to that of patients without cancer (Sakanoue et al., Int. J. Cancer 48:803, 1991).
Increased tumorigenicity is also correlated with overexpression of PKC in cultured cells inoculated into nude mice. A mutant form of PKC induces highly malignant tumor cells with increased metastatic potential. Sphingosine and related inhibitors of PKC activity have been shown to inhibit tumor cell growth and radiation-induced transformation in vivo (Endo et al., Cancer Research 51:1613, 1991; Borek et al., Proc. Natl. Acad. Sci. (USA) 88:1953, 1991). A number of experimental or clinically useful anti-cancer drugs show modulatory effects on PKC. Therefore, inhibitors of PKC may be important cancer-preventive or therapeutic agents. PKC has been suggested as a plausible target for more rational design of conventional anti-cancer drugs (Gescher, A. and Dale, I. L., Anti-Cancer Drug Design, 4:93, 1989).
Experiments also indicate that PKC plays an important role in the pathophysiology of hyperproliferative skin disorders such as psoriasis and skin cancer. Psoriasis is characterized by inflammation, hyperproliferation of the epidermis and decreased differentiation of cells. Various studies indicate a role for PKC in causing these symptoms. PKC stimulation in cultured keratinocytes can be shown to cause hyperproliferation. Inflammation can be induced by phorbol esters and is regulated by PKC. DAG is implicated in the involvement of PKC in dermatological diseases, and is formed to an increased extent in psoriatic lesions.
Inhibitors of PKC have been shown to have both antiproliferative and antiinflammatory effects in vitro. Some antipsoriasis drugs, such as cyclosporine A and anthralin, have been shown to inhibit PKC. Inhibition of PKC has been suggested as a therapeutic approach to the treatment of psoriasis (Hegemann, L. and G. Mahrle, Pharmacology of the Skin, H. Mukhtar, ed., pp. 357-368, CRC Press, Boca Raton, Fla., 1992).
The oligonucleotides of the invention are useful in the therapeutic treatment of diseases associated with PKC isoforms. Such diseases include, but are not limited to, hyperproliferative and inflammatory conditions including psoriasis, tumors and cancers, for example glioblastoma, bladder cancer, breast cancer, lung cancer and colon cancer.
Although numerous compounds have been identified as PKC inhibitors (see Hidaka and Hagiwara, Trends in Pharm. Sci. 8:162, 1987, for a review), few have been found which inhibit PKC specifically. While the quinoline sulfonamide derivatives such as 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) inhibit PKC at micromolar concentrations, they exhibit similar enzyme inhibition kinetics for PKC and the CAMP-dependent and cGMP-dependent protein kinases. Staurosporine, an alkaloid product of Streptomyces sp., and its analogs, are potent in vitro inhibitors of PKC. However, these drugs exhibit only limited selectivity among different protein kinases (Gescher, Anti-Cancer Drug Design 4:93, 1989). Certain ceramides and sphingosine derivatives have been shown to have PKC inhibitory activity and to have promise for therapeutic uses, however, there remains a long-felt need for specific inhibitors of the enzymes.
There is also a desire to inhibit specific PKC isozymes, both as a research tool and in diagnosis and treatment of diseases which may be associated with particular isozymes. Godson et al. (J. Biol. Chem. 268:11946, 1993) disclose the stable transfection of antisense PKC-xcex1 cDNA in cytomegalovirus promotor-based expression vectors to specifically decrease expression of PKC-xcex1 protein by approximately 70%. It was demonstrated that this inhibition caused a loss of phospholipase A2-mediated arachidonic acid release in response to the phorbol ester PMA. Attempts by the same researchers at inhibiting PKC activity with oligodeoxynucleotides were ultimately unsuccessful. Ahmad et al. (Neurosurg. 35:904, 1994) disclose that transfection of the human glioblastoma cell line, U-87, with vectors expressing antisense RNA to PKC-xcex1 inhibits growth of the glioblastoma cells in vitro and in vivo. Diaz-Meco Conde et al. (WO Application 93/20101, published Oct. 14, 1993) disclose a peptide corresponding to the pseudo-substrate region of PKC-xcex6 and oligonucleotides antisense to this isozyme. Alvaro et al. (WO Application 94/29455, published Dec. 22, 1994) have identified a novel mutant form of PKC associated with tumors and disclose oligonucleotide sequences complementary to the mutant form.
The oligonucleotides of the invention are designed to be targeted to and modulate, as defined herein, the xcex2I, xcex2II, xcex3, xcex4, xcex5, xcex6 or xcex7 isoforms of PKC. Accordingly, the oligonucleotides of the invention are useful for the inhibition of specific non-xcex1 PKC isozymes, either in the course of research or in the diagnosis and treatment of diseases associated with particular non-xcex1 PKC isozymes.
2. Oligonucleotides Having Phosphorothioate Linkages of High Chiral Purity
Oligonucleotides are known to hybridize to single-stranded RNA or single-stranded DNA. Hybridization is the sequence-specific base pair hydrogen bonding of bases of the oligonucleotides to bases of target RNA or DNA. Such base pairs are said to be complementary to one another.
In determining the extent of hybridization of an oligonucleotide to a complementary nucleic acid, the relative ability of an oligonucleotide to bind to the complementary nucleic acid may be compared by determining the melting temperature of a particular hybridization complex. The melting temperature (Tm), a characteristic physical property of double helices, denotes the temperature (xc2x0 C.) at which 50% helical (hybridized) versus coil (unhybridized) forms are present. Tm is measured by using the UV spectrum to determine the formation and breakdown (melting) of the hybridization complex. Base stacking which occurs during hybridization, is accompanied by a reduction in UV absorption (hypochromicity). Consequently, a reduction in UV absorption indicates a higher Tm. The higher the Tm, the greater the strength of the bonds between the strands.
Oligonucleotides can be used to effect enzymatic cleavage of a target RNA by using the intracellular enzyme RNase H. The mechanism of such RNase H cleavage requires that a 2xe2x80x2-deoxyribofuranosyl oligonucleotide hybridize to a target RNA. The resulting DNA-RNA duplex activates the RNase H enzyme and the activated enzyme cleaves the RNA strand. Cleavage of the RNA strand destroys the normal function of the target RNA. Phosphorothioate oligonucleotides operate via this type of mechanism. However, for a DNA oligonucleotide to be useful for cellular activation of RNase H, the oligonucleotide must be reasonably stable to nucleases in order to survive in a cell for a time period sufficient for RNase H activation. For non-cellular uses, such as use of oligonucleotides as research reagents, such nuclease stability may not be necessary.
Several publications of Walder et al. describe the interaction of RNase H and oligonucleotides. Of particular interest are: (1) Dagle et al., Nucleic Acids Research 18:4751, 1990; (2) Dagle et al., Antisense Research And Development 1:11, 1991; (3) Eder et al., J. Biol. Chem. 266:6472, 1991; and (4) Dagle et al., Nucleic Acids Research 19:1805, 1991. According to these publications, DNA oligonucleotides having both unmodified phosphodiester internucleoside linkages and modified phosphorothioate internucleoside linkages are substrates for cellular RNase H. Since they are substrates, they activate the cleavage of target RNA by RNase H. However, the authors further note that in Xenopus embryos, both phosphodiester linkages and phosphorothioate linkages are also subject to exonuclease degradation. Such nuclease degradation is detrimental since it rapidly depletes the oligonucleotide available for RNase H activation.
As described in the above references (1), (2) and (4), to stabilize oligonucleotides against nuclease degradation while still providing for RNase H activation, 2xe2x80x2-deoxy oligonucleotides having a short section of phosphodiester linked nucleotides positioned between sections of phosphoramidate, alkyl phosphonate or phosphotriester linkages were constructed. While the phosphoamidate-containing oligonucleotides were stabilized against exonucleases, in reference (4) the authors noted that each phosphoramidate linkage resulted in a loss of 1.6xc2x0 C. in the measured Tm value of the phosphoramidate containing oligonucleotides. Such a decrease in the Tm value is indicative of a decrease in hybridization between the oligonucleotide and its target nucleic acid strand.
Applications of oligonucleotides as diagnostics, research reagents, and therapeutic agents require that the oligonucleotides be transported across cell membranes or taken up by cells, appropriately hybridize to target RNA or DNA, and subsequently terminate or disrupt target nucleic acid function. These critical functions depend partly on the initial stability of oligonucleotides towards nuclease degradation. Further, these functions depend on specificity of the oligonucleotide for a target DNA or RNA molecule.
A serious deficiency of oligonucleotides for these purposes is their susceptibility to enzymatic degradation by a variety of ubiquitous nucleases which may be intracellularly and extracellularly located. Unmodified, xe2x80x9cwild typexe2x80x9d, oligonucleotides are not useful as therapeutic agents because they are rapidly degraded by nucleases. Therefore, modification of oligonucleotides for conferring nuclease resistance on them has been the primary focus of research directed towards the development of oligonucleotide therapeutics and diagnostics.
Modifications of oligonucleotides to enhance nuclease resistance has generally taken place on the sugar-phosphate backbone, particularly on the phosphorous atom. Phosphorothioates have been reported to exhibit resistance to nucleases. In addition, phosphorothioate oligonucleotides are generally more chemically stable than natural phosphodiester oligonucleotides. Phosphorothioate oligonucleotides also exhibit solubility in aqueous media. Further, phosphorothioate oligonucleotide-RNA heteroduplexes can serve as substrates for endogenous RNase H. Additionally, phosphorothioate oligonucleotides exhibit high thermodynamic stability. However, while the ability of an oligonucleotide to bind to a target DNA or RNA with fidelity is critical for its hybridization to the target DNA or RNA, modifications at the phosphorous atom of the oligonucleotides, while exhibiting various degrees of nuclease resistance, have generally suffered from inferior hybridization properties (Cohen, J. S., Ed., Oligonucleotides: Antisense Inhibitors of Gene Expression, CRC Press, Inc., Boca Raton, Fla., 1989).
One reason for this inferior hybridization may be the prochiral nature of the phosphorous atom. Modifications on the internal phosphorous atom of modified phosphorous oligonucleotides results in Rp and Sp stereoisomers. Modified phosphorus oligonucleotides obtained thus far, wherein the resulting molecule has nonsymmetrical substituents, have been racemic mixtures having 2n isomers, with n equal to the number of phosphorothioate intersugar linkages in the oligonucleotide. Thus, a 15-mer phosphorothioate oligonucleotide, containing 14 asymmetric centers has 214 or 16,384 diastereomers. In view of this, in a racemic mixture, only a small percentage of the oligonucleotides are likely to specifically hybridize to a target mRNA or DNA with optimal affinity.
Chemically synthesized phosphorothioate oligonucleotides having chirally pure intersugar linkages had thus far been limited to molecules having only one or two diastereomeric intersugar linkages. Until recently, the effects of induced chirality in chemically synthesized racemic mixtures of sequence-specific phosphorothioate oligonucleotides had not been assessed, since synthesis of oligonucleotides having chirally pure intersugar linkages had yet to be accomplished by automated synthesis. This was due to the non-stereospecific incorporation of sulfur during automated synthesis. For example, Stec et al. (J. Chromatography 326:263, 1985), synthesized certain oligonucleotide phosphorothioates having racemic intersugar linkages, however, they were able to resolve only the diastereomers of certain small oligomers having one or, at most, two diastereomeric phosphorous intersugar linkages. Stec et al. subsequently reported (Nucl. Acids Res. 19:5883, 1991)the automated stereocontrolled synthesis of oligonucleotides. The procedure described in the above-mentioned reference utilizes base-catalyzed nucleophilic substitution at a pentavalent phosphorothioyl center.
The synthesis of phosphorothioates having all Rp intersugar linkages using enzymatic methods has been investigated by several authors (Burgers and Eckstein, J. Biol. Chem. 254:6889, 1979); Gupta et al., J. Biol. Chem. 256:7689, 1982); Brody and Frey, Biochemistry 20:1245, 1981); and Eckstein and Jovin, Biochemistry 2:4546, 1983). Brody et al. (Biochemistry 21:2570, 1982) and Romaniuk and Eckstein, (J. Biol. Chem. 257:7684, 1982) enzymatically synthesized poly TpA and poly ApT phosphorothioates, while Burgers and Eckstein (Proc. Natl. Acad. Sci. (USA) 75:4798, 1978) enzymatically synthesized poly UpA phosphorothioates. Cruse et al. (J. Mol. Biol. 192:891, 1986) linked three diastereomeric Rp GpC phosphorothioate dimers via natural phosphodiester bonds into a hexamer.
The relative ability of an oligonucleotide to bind to complementary nucleic acids may be compared by determining the melting temperature of a particular hybridization complex. The melting temperature (Tm), a characteristic physical property of double helixes, denotes the temperature (xc2x0 C.) at which 50% helical versus coil (unhybridized) forms are present. Tm is measured by using the UV spectrum to determine the formation and breakdown (melting) of hybridization. Base stacking which occurs during hybridization, is accompanied by a reduction in UV absorption (hypochromicity). Consequently a reduction in UV absorption indicates a higher Tm. The higher the Tm, the greater the strength of the binding of the strands. Non-Watson-Crick base pairing has a strong destabilizing effect on the Tm.
In a preliminary report (Stec, J. W., Oligonucleotides as Antisense Inhibitors of Gene Expression: Therapeutic Implications, Meeting Abstracts, Jun. 18-21, 1989), thymidine homopolymer octamers having all but one linkage being modified phosphate linkages (xe2x80x9call except onexe2x80x9d) Rp stereoconfiguration or xe2x80x9call except onexe2x80x9d Sp stereoconfiguration in the intersugar linkages were formed from two thymidine methylphosphonate tetrameric diastereomers linked by a natural phosphodiester bond. It was noted that a Rp xe2x80x9call except onexe2x80x9d methylphosphonate non-sequence-specific thymidine homooctamer, i.e., (dT)8 having all but one Rp intersugar linkage, formed a thermodynamically more stable hybrid (Tm 38xc2x0 C.) with a 15-mer deoxyadenosine homopolymer (i.e., (dA)15) than a hybrid formed by a similar thymidine homopolymer having xe2x80x9call except onexe2x80x9d Sp configuration methylphosphonate linkages and of d(A)15 (Tm less than 0xc2x0 C.), i.e., a d(T)15 having all but one Sp intersugar linkage. A hybrid between (dT)8 having natural phosphodiester linkages (i.e., octathymidylic acid) and d(A)15 was reported to have a Tm of 14xc2x0 C.
More recently, Ueda et al. (Nucl. Acids Research 19:547, 1991) enzymatically synthesized mRNAs intermittently incorporating Rp diastereomeric phosphorothioate linkages for use in translation systems. Ueda et al. employed T7 coliphane DNA having seventeen promoters and one termination site for T7 RNA polymerase. In vitro synthesis by T7 RNA polymerase produced mRNAs having from several hundred to tens of thousands of nucleotides.
Backbone chirality may also affect the susceptibility of a phosphorothioate oligonucleotide-RNA heteroduplex to RNase H activity. The ability to serve as a template for RNAse H has significant therapeutic implications since it has been suggested that RNAse H causes cleavage of the RNA component in an RNA-DNA oligonucleotide heteroduplex. With oligonucleotides containing racemic mixtures of Rp and Sp intersugar linkages, it is not known if all phosphorothioate oligonucleotides can function equally as substrates for RNase H. For a variety of catalytic reactions, hydrolysis of the phosphodiester backbone of nucleic acids proceeds by a stereospecific mechanism (an in-line mechanism) and inversion of configuration. Therefore, there may be only a small percentage of oligonucleotides in a racemic mixture that contain the correct chirality for maximum hybridization efficiency and termination of translation. Thus, increasing the percentage of phosphorothioate oligonucleotides that can serve as substrates for RNAse H in a heteroduplex will likely lead to a more efficacious compound for antisense and other oligonucleotide therapies.
To enhance hybridization fidelity, phosphorothioate oligonucleotides having substantially chirally pure intersugar linkages are greatly desired. Further, such phosphorothioate oligonucleotides having substantially chirally pure intersugar linkages would lead to more efficacious therapeutic compounds. However, until now little success has been achieved in synthesizing such molecules. Therefore, simple methods of synthesizing phosphorothioate oligonucleotides having substantially chirally pure intersugar linkages are greatly desired.
Current agents which modulate the activity or metabolism of protein kinase C (PKC) either exhibit many unacceptable side effects due to their lack of specificity for a particular isozyme, or exhibit only limited effectiveness in inhibiting PKC. The instant invention circumvents problems encountered by prior workers by modulating the production of a specific PKC isozyme, rather than inhibiting PKC isozymes generally and directly, to achieve the therapeutic effect. In the instant invention, the oligonucleotide is designed to bind directly to mRNA or to a gene encoding a PKC isozyme, ultimately modulating the amount of the PKC isozyme made from the corresponding gene. It is an object of this invention to provide sequence-specific phosphorothioate oligonucleotides having substantially chirally pure (either all Rp or all Sp) intersugar linkages, wherein such oligonucleotides modulate the expression of the xcex2I, xcex2II, xcex3, xcex4, xcex5, xcex6 or xcex7 isoform of protein kinase C.
It is another object of this invention to provide methods for synthesis of sequence-specific phosphorothioate oligonucleotides having substantially chirally pure intersugar linkages, wherein such olgonucleotides modulate the expression of a xcex2I, xcex2II, xcex3, xcex4, xcex5, xcex6 or xcex7 isoform of protein kinase C.
These and other objects of the present invention shall become apparent to persons skilled in the art to which this invention pertains given this specification and the claims appended hereto.